Microplate-based Method Research Articles

Applicability of enzymatic and phenol–sulfuric acid methods for determination of cellulose nanofibers in ecotoxicity testing

In parallel with the development of novel materials, such as cellulose nanofibers (CNFs), their effects on aquatic organisms should be evaluated. In ecotoxicity testing, concentration measurements are important to obtain accurate exposure concentrations; however, few studies have measured the concentration and dispersion state of CNFs in test solutions. Therefore, we evaluated the applicability of enzymatic and phenol–sulfuric acid methods for CNF quantification in ecotoxicity testing. In the enzymatic method, CNF content was determined by quantifying the d-glucose produced from the CNFs using cellulase with a glucose assay kit. Three types of cellulases were purchased, and impurities such as d-glucose were identified and removed. The differences in CNF degradation efficiency among the cellulases were evaluated, and a cellulase suitable for CNF degradation was selected. In the phenol–sulfuric acid method, a microplate-based method was employed to handle a large number of samples. We demonstrated that both the enzymatic and phenol–sulfuric acid methods could quantify CNFs in media for ecotoxicity testing. The enzymatic method was able to measure lower CNF concentrations (~ 1 mg/L) than the phenol–sulfuric acid method (~ 10 mg/L) and had less interference with CNF quantification by algae and non-cellulosic polysaccharides.

Development of a microplate-based novel toxicity bioassay using Chlorophyta and Phaeophyceae macroalgae.

Macroalgae are one of the main producers in marine environments. However, only a few toxicity test methods have been established that use reference strains of macroalgae to evaluate the effects of chemicals on the growth and reproduction of macroalgae to monitor water quality. We selected reference strains of Chlorophyta, Ulva aragoënsis; Phaeophyceae, Ectocarpus siliculosus; and wakame, Undaria pinnatifida, as test species to establish a microplate-based method to investigate the toxicity of potassium dichromate, 3,5-dichlorophenol, and two common herbicides (diuron and simazine). We determined the growth of the three macroalgae in their early life stages and during the sporangia formation stage in E. siliculosus under laboratory conditions. We observed that the growth and sporangia formation in these algae were impaired in a dose-dependent manner. Additionally, we investigated the sensitivity of these macroalgae by comparing the toxicity values of toxicants used in this study with those obtained from a database. Compared to other microalgae and plant species, macroalgae showed a relatively high sensitivity to organic compounds, including herbicides. Growth tests using U. aragoënsis and E. siliculosus produced reliable results at 0-32 and 25-32 practical salinity units (PSU), respectively. The tests established in this study could test the toxicity of chemical substances in macroalgae and are thus expected to contribute to a better understanding of the environmental risks of chemical substances on aquatic biota. The tests could be applied to all effluent toxicity tests used for the management of seawater and brackish water quality.

Enhancement of chlorhexidine activity against planktonic and biofilm forms of oral streptococci by two Croton spp. essential oils from the Caatinga biome

This work investigates the ability of two Croton spp. essential oils (EO) to enhance chlorhexidine (CHX) activity against oral streptococci. EO’s chemical composition of Croton argyrophyllus and C. pluriglandulosus was determined by GC-MS/FID. The microbial growth kinetics and minimum inhibitory concentration (MIC) of EOs and CHX were determined, followed by their synergism against S. mutans UA159 and ATCC 25175, S. salivarius ATCC 7073 and S. sp. ATCC 15300. The microplate-based method was used to determine the EO/CHX activity against 24-h-old biofilms. The major compounds were α-pinene (54.74%) and bicyclogermacrene (16.08%) for EOAr and 1,8-cineole (17.41%), methyleugenol (16.06%) and elemicin (15.99%) for EOPg. Both EO had MIC around 16,000 µg/mL. EOs/CHX presented a synergistic effect against most strains (FICi from 0.133 to 0.375), and OE/CHX-treated biofilms showed a reduction in biomass and cell viability compared to CHX, only (p < 0.01). Thus, the EOs works as natural adjuvants for CHX.

An optimized microplate-based method to evaluate complement-dependent hemolysis mediated by intravenous immunoglobulins (IVIG)

Hemolytic reactions can cause serious complications after administration of Intravenous Immunoglobulin (IVIG), due to passive transfer of anti-A and anti-B IgG antibodies (isoagglutinins). A maximum allowable amount of isoagglutinins is established in the US and EU for licensed IVIG, as measured by a specified direct hemagglutination test (DHAT). Despite this limit, reports of hemolysis have increased over time, raising the question of how well the DHAT predicts clinically significant hemolysis. This study was undertaken to develop a microplate-based complement-dependent hemolysis assay (CDHA) that reproducibly measures functional hemolytic activity of IVIG, for assessment of IVIG products. An IVIG working reference reagent (NIBSC 14/160) was qualified as an assay control and for quantitation purposes. Hemolytic activities of 36 IVIG product lots encompassing seven brands and including 6 clinically hemolytic lots were measured. Hemolytic activity varied among IVIG product brands, and to a lesser extent, from lot-to-lot for individual brands. Correlation between the CDHA and DHAT was not robust which may reflect imprecision of the DHAT method or additional variables that influence complement-dependent hemolysis after opsonization. In conclusion, the CDHA provides a simple, specific, and sensitive tool for IVIG product characterization and investigation of hemolytic events by manufacturers, researchers, and regulatory authorities.

A Microplate-Based Approach to Map Interactions between TDP-43 and α-Synuclein.

Trans-active response DNA-binding protein (TDP-43) is a multifunctional regulatory protein, whose abnormal deposition in neurons was linked to debilitating neurodegenerative diseases, such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Limbic-predominant age-related TDP-43 encephalopathy, and Alzheimer’s disease with a secondary pathology. Several reports showed that TDP-43 proteinopathy as a comorbidity can form aggregates with other pathological proteins. The co-deposition of alpha synuclein and TDP-43 inclusions was previously reported in glial cells and by observing TDP-43 proteinopathy in Lewy body disease. In this study, it was hypothesized that alpha synuclein and TDP-43 may co-aggregate, resulting in comorbid synucleinopathy and TDP-43 proteinopathy. A solid-phase microplate-based immunoassay was used to map out the epitopes of anti-TDP-43 antibodies and locate the interaction of TDP-43 with α-synuclein. A region of the low complexity domain of TDP-43 (aa 311–314) was shown to interact with full-length α-synuclein. Conversely, full-length TDP-43 was shown to bind to the non-amyloid beta component of α-synuclein. Using in silico sequence-based prediction, the affinity and dissociation constant of full-length TDP-43 and α-synuclein were calculated to be −10.83 kcal/mol and 1.13 × 10−8, respectively. Taken together, this microplate-based method is convenient, economical, and rapid in locating antibody epitopes as well as interaction sites of two proteins.

Multivariable optimization of ultrasound-assisted extraction for the determination of phenolic and antioxidants compounds from arrayan (Luma apiculata (DC.) Burret) leaves by microplate-based methods and mass spectrometry

The present study reports a multivariable optimization of ultrasound-assisted extraction of phenolic compounds from arrayan leaves. The parameters ethanol percentage in extraction solvent, temperature and extraction time, and mass to solvent ratio were optimized applying full factorial and central composite designs. According to the models, the optimal extraction conditions were: 42% of ethanol, extraction time of 27 min, extraction temperature of 50ºC and a mass to solvent ratio of 1:33.4 g mL−1. Under these conditions, a total phenolic content (TPC) of 128.16 ± 1.18 and 593.64 ± 6.49 mg of gallic acid equivalent per g of dry weight (DW) were determined in raw extract (RE) and polyphenols-rich extract (PRE), which was obtained by semi-preparative chromatography on XAD-7 column. Antioxidant capacity determined via FRAP analysis showed values of 1349.53 ± 28.99 and 6175.47 ± 127.64 µmol Trolox equivalent per g of DW for RE and PRE, and DPPH IC50 values of 831.40 ± 0.80 and 132.21 ± 2.51 µg mL−1, respectively. Polyphenols profile analyzed by liquid chromatography-mass spectrometry showed the presence of quercetin 3-ß-D-glucoside, myricetin, quercetin, kaempferol 3-glucoside and gallic acid; the latter compounds are reported for the first time in arrayan leaves. Due to the presence of polyphenols, antiviral activity was assayed over norovirus and hepatitis A showing a significant dose-dependent effect.

Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture

BackgroundQuantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design.ResultsWe present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail.ConclusionsThe method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

Recombinant Bacteriophage Cell-Binding Domain Proteins for Broad-Spectrum Recognition of Methicillin-Resistant Staphylococcus aureus Strains.

Methicillin-resistant Staphylococcus aureus (MRSA) has been well-recognized as one of the most common multiresistant bacteria threatening human health. Broad-spectrum recognition of multiple MRSA strains can meet the urgent demands for efficient diagnosis and subsequent decision of relevant treatment of MRSA-induced infections. Here, recombinant cell-binding domain (CBD) and green fluorescent protein-fused CBD of MRSA bacteriophage were expressed in soluble form. Distinct from the strain-specific MRSA bacteriophage, both recombinant CBD proteins displayed broad-spectrum recognition capability toward all five staphylococcal cassette chromosome mec types of MRSA. Furthermore, they did not display any lytic activity toward the host bacteria, which facilitated the capture of whole MRSA cells with ideal flexibility for downstream manipulation and tracing. For demonstration of their application potential, a flow cytometry method employing the recombinant CBD proteins as the recognition agents was established to detect MRSA within a dynamic range of 1.5 × 102 to 1.5 × 106 cfu mL-1. The method can exclude potential interference from methicillin-sensitive Staphylococcus aureus strains and other bacterial species. The recombinant CBD proteins were also successfully employed in antibiotic susceptibility testing of MRSA with a microplate-based method. The obtained results were consistent with those by the standard broth microdilution method. The satisfying results demonstrated their great application potential in clinical diagnosis and treatment of MRSA-induced infections.

Miniaturized culture method for the statistical study of growth rate and carbohydrate content of Picochlorum sp. D8 isolated from the Persian Gulf

The Microplate-based method is a useful means for high-throughput screening and kinetic studies of microalgal growth. In this study, the statistical evaluation of growth and carbohydrate production by a marine microalga, designated as Picochlorum sp. D8, which is taxonomically affiliated to Picochlorum sp. SENEW3 was done on a miniaturized scale. Initially, the significance of various factors was studied by using the Plackett–Burman Design. The Central Composite Design was applied to compute the optimum level of significant factors, e.g., phosphate (KH2PO4+K2HPO4) and FeCl3·6H2O. Maximum carbohydrate production was obtained at low phosphate (0.148 g/L) and FeCl3·6H2O (0.009 g/L) concentrations. The validation experiments were carried out under optimal conditions, where carbohydrate content increased by 3.5 times. The analyses of the results showed a quadratic effect of phosphate and FeCl3·6H2O concentrations.

Proliferation of enterotoxigenic Escherichia coli strain TW11681 in stools of experimentally infected human volunteers

BackgroundAs part of the effort to develop an enterotoxigenic Escherichia coli (ETEC) human challenge model for testing new heat-stable toxin (ST)-based vaccine candidates, a controlled human infection model study based on the ST-producing ETEC strain TW11681 was undertaken. Here, we estimate stool TW11681 DNA concentration and evaluate its association with dose, clinical symptoms, and with levels of antibodies targeting the CfaB subunit of the ETEC Colonization Factor Antigen I and the E. coli mucinase YghJ. Nine volunteers ingested different doses of the strain and were subsequently followed for 9 days with daily stool specimen collection and clinical examination. Stool DNA was purified by using a newly developed microplate-based method, and DNA originating from TW11681 was quantified by using a probe-based quantitative PCR assay. Antibody levels against CfaB and YghJ were measured in serum collected before and 10 and 28 days after TW11681 was ingested by using a bead-based flow cytometry immunoassay.ResultsFor 6 of the 9 volunteers, the stool TW11681 DNA concentration increased sharply a median 3.5 (range 2–5) days after dose ingestion, peaking at a median of 5.4% (range 3.3–8.2%) of the total DNA in the specimen. The concentration then fell sharply during the subsequent days, sometimes even before the onset of antibiotic treatment. The size or timing of these proliferation peaks did not seem to be associated with the number of TW11681 bacteria ingested, but the 2 volunteers who developed diarrhea and all five who experienced abdominal pains or cramps had these peaks. The 3 volunteers who did not have the proliferation peaks experienced fewer symptoms and they generally had relatively low CfaB- and YghJ-specific antibody levels before ingesting the strain and subsequently weaker responses than the other volunteers afterwards.ConclusionsSince the lack of proliferation peaks appears to be associated with fewer clinical symptoms and lower serum antibody responses to virulence factors of the infecting strain, it may be important to account for proliferation peaks when explaining results from controlled human infection model studies and for improving the accuracy of protective efficacy estimates when testing new ETEC diarrhea vaccine candidates.

Applicability and reliability of the glucose oxidase method in assessing α-amylase activity

Glucose oxidase (GOD) is an enzyme widely used in glucose monitoring systems owing to its high specificity towards glucose. However, in our previous work maltose was found to show significant interaction with GOD and based on this observation, a novel microplate-based method was developed to assess α-amylase inhibitory activity (GOD method). Concerns regarding the interaction of GOD with maltose has limited the widespread use of the GOD method in assessing α-amylase activity. The present paper provides answers to concerns regarding the interaction of GOD with maltose using HPLC studies and application of the GOD method in assessing α-amylase activity. According to the results, the newly developed GOD method can be considered as a well-suited method for the determination of α-amylase activity and as an easy method to do kinetic studies compared to other available methods.

Glutathione Peroxidase Activity of Biological Samples Using A Novel Microplate-Based Method

Background: Glutathione peroxidase (GSH-Px) is one of the major enzyme in the antioxidative defense mechanism present in the cells. Methods: In the present study a novel and simple microplate-based method was developed, using the cupric neocuproine complex (Cu(Nc)2 2+) as a chromogenic oxidizing reagent, for the assessment of GSH-Px activity of biological samples for the first time. In this GSH-Px enzyme activity measurement, the GSH-Px catalyzed oxidation of Reduced Glutathione (GSH) gives rise to oxidized form of GSH (GSSG). The recommended method was based on the reduction of Cu(Nc)2 2+ to highly colored Cu(I)- neocuproine complex (Cu(Nc)2 +) by the unconsumed GSH, and measurement spectrophotometrically at 450 nm, the difference being correlated to GSH-Px activity of the analytes. Results: Under the optimum conditions, a linear calibration graph was obtained in the range of 18.5- 92.5 µM of GSH with Limit Of Detection (LOD) of 1.03 µM. Application of developed method to tissue homogenates (n = 9) provided GSH-Px activity values in agreement with those of the reference GSH-Px-5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) method. Conclusion: Cupric Ion Reducing Antioxidant Capacity (CUPRAC) antioxidant method was implemented in a microformat (96 well plates) and the reaction time of original CUPRAC method was significantly shortened from 30 to 4 min. Multi-sample can be simultaneously detected thanks to a novel microplate method. Keywords: GSH-Px activity, microplate based method, modified CUPRAC method, biological samples, neocuproine complex, reduced glutathione (GSH).

Cross-laboratory comparison of fluorimetric microplate and colorimetric bench-scale soil enzyme assays

There has been growing interest in fluorescence-based microplate methods to measure enzyme activities due to the sensitivity of fluorimetric detection and the potential for simultaneous and rapid assaying of multiple enzyme activities in the same soil suspension. However, micro-scale methods could introduce considerable operator error such as: 1) the requirement to put soil samples into a suspension; 2) the very small amounts of soil placed in each microplate well; 3) pipetting error because μL volumes are required; and 4) the need for standard curve calibration with every sample to account for quenching. For valid data comparison and interpretation, there is clearly a need to have a strict and agreed-upon enzyme assay protocol to standardize the microplate-based method. Therefore, the objectives were to: 1) determine the reproducibility and comparability of the standard p-nitrophenol bench-scale and 4-methylumbelliferone microplate enzyme assays measured by five laboratories for β-glucosidase (EC 3.2.1.21) and acid phosphomonoesterase (EC 3.1.3.2) on the same soil samples; and 2) determine the degree and the sources of variability associated with the assays within and among the laboratories. The results showed that overall variability was highest for replication on the microplate (n = 4), whereas suspension replication had low CVs. This suggests an important source of variation is from pipetting not variability from soil suspensions. A major effort was made to control for methodological differences by using air-dried soils (therefore more stable over varying storage periods) and operator consistency for each task across the labs (e.g. preheated reagents, microplate reader sensitivity set to the highest standard, readings taken within an hour of reaction termination, and controls for substrate autohydrolysis). As a result, the differences among labs were much smaller than differences due to soil type for the microplate method, indicating operator error can be minimized by following the same strict protocol. At the molar level, enzyme activity rates measured across the five labs were not the same between bench and MUF microplate methods (although they were within an order of magnitude), but were quite similar in terms of ranking of soil management treatments and soil types (Table 2). Correlations between bench and microplate assays were strong for both enzymes, although slightly stronger for acid phosphomonoesterase (r = 0.93) than β-glucosidase (r = 0.81). Additionally, for both acid phosphomonoesterase and β-glucosidase, correlation r values were mostly similar for MUF microplate and PNP bench method correlation with EL-FAME biomarkers, suggesting both methods were measuring activity originating from the same microbial groups. We conclude that different labs using the same MUF microplate protocol tested, gives reasonably similar absolute activity values, variability, and ranking of treatments (highest to lowest). We propose that the MUF microplate method described in this study be considered as a standard protocol for assaying soil enzyme activities, providing that the buffer pH for the incubation be adjusted to the optimal pH according to the enzyme of interest.

Robust Microplate-Based Methods for Culturing and in Vivo Phenotypic Screening of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii (Cr), a unicellular alga, is routinely utilized to study photosynthetic biochemistry, ciliary motility, and cellular reproduction. Its minimal culture requirements, unicellular morphology, and ease of transformation have made it a popular model system. Despite its relatively slow doubling time, compared with many bacteria, it is an ideal eukaryotic system for microplate-based studies utilizing either, or both, absorbance as well as fluorescence assays. Such microplate assays are powerful tools for researchers in the areas of toxicology, pharmacology, chemical genetics, biotechnology, and more. However, while microplate-based assays are valuable tools for screening biological systems, these methodologies can significantly alter the conditions in which the organisms are cultured and their subsequent physiology or morphology. Herein we describe a novel method for the microplate culture and in vivo phenotypic analysis of growth, viability, and photosynthetic pigments of C. reinhardtii. We evaluated the utility of our assay by screening silver nanoparticles for their effects on growth and viability. These methods are amenable to a wide assortment of studies and present a significant advancement in the methodologies available for research involving this model organism.

Microplate-Based Method for High-Throughput Screening (HTS) of Chromatographic Conditions Studies for Recombinant Protein Purification.

High-throughput screening (HTS) systems have emerged as important tools to provide fast and low cost evaluation ofseveral conditions at once since it requires small quantities of material and sample volumes. These characteristics are extremely valuable for experiments with large number of variables enabling the application of design of experiments (DoE) strategies or simple experimental planning approaches. Once, the capacity of HTS systems to mimic chromatographic purification steps was established, several studies were performed successfully including scale down purification. Here, we propose a method for studying different purification conditions that can be used for any recombinant protein, including complex and glycosylated proteins, using low binding filter microplates.

NADPH oxidase activity: Spectrophotometric determination of superoxide using pyrogallol red

A simple and fast spectrophotometric methodology able to quantify superoxide released by NADPH oxidase from differentiated promyelocytic leukaemia (HL-60) cells using pyrogallol red is described.The latter is based on the known stoichiometry of the reaction between superoxide and pyrogallol red and the inability of pyrogallol red to react with hydrogen peroxide. In addition, we developed a 96-wells microplate-based method able to determine NADPH oxidase activity. Using this method, we determined pharmacological properties of the NADPH oxidase inhibitors VAS2870 and diphenyleneiodonium and the obtained IC50 values were in good agreement with previous reported data. NOX2 is highly expressed in differentiated promyelocytic leukaemia cells, whereas other isoforms are not detected or expressed at low amounts. Likewise, this methodology may be a useful assay for NOX2 inhibitor screening. NADPH oxidases are involved in several physiological and pathological processes, rendering its pharmacological modulation an attractive research target. In this context, this simple assay can be used for NADPH oxidase inhibitor screening as well as aiding in the study of different biological conditions that involve NADPH oxidase activity.

Laboratory analysis of soil respiration using oxygen-sensitive microplates

Soil respiration is usually monitored by measuring CO2 efflux. Most techniques available for such kind of analyses are inconvenient and difficult to adapt to micro-method format. The present study evaluates a new microplate-based method for studying soil respiration in the laboratory. Second-generation oxygen-sensitive microplates (OSM - containing a fluorescent probe attached to the bottom of the well which provides time-resolved fluorescence data) were used to measure soil respiration either in microcosm assays or in soil water extracts. The latter procedure (water extracts) was the least cumbersome, hence it was selected for further experiments. Soil respiration curves generally showed no lag phase, starting with an exponential oxygen consumption phase, followed by a period where respiration became stable after 8–10h of incubation at 25°C. Once the procedure for measurement of oxygen consumption in soil was established, the acute toxic effect of diverse chemicals on soil was analysed with OSM. Streptomycin and penicillin failed to reduce soil respiration. Kanamycin plus neomycin, trimethoprim and 5-fluorocytosine exhibited limited inhibitory effects. In contrast, some chemicals (copper sulphate and amphotericin B) and fungicides (such as dodine and fosetyl) noticeably reduced fluorescence readings, showing concentrations to give half-maximal inhibition of respiration (ICR50) ranging from 0.0064 to 0.082g/L. Finally, some insecticides and soil amendments assayed were either neutral or increased respiration.It is concluded that OSM are reliable, convenient, and yield quantitative results. Moreover, the system is relatively inexpensive and amenable to automation. However, results obtained using soil water extracts may be different from those derived from undisturbed soil aggregates, clods or slurries studied under field conditions.

Ultrasensitive and high-throughput analysis of chlorophyll a in marine phytoplankton extracts using a fluorescence microplate reader.

Chlorophyll a (Chl a) is the predominant pigment in every single photosynthesizing organism including phytoplankton and one of the most commonly measured water quality parameters. Various methods are available for Chl a analysis, but the majority of them are of limited throughput and require considerable effort and time from the operator. The present study describes a high-throughput, microplate-based fluorometric assay for rapid quantification of Chl a in phytoplankton extracts. Microplate sealing combined with ice cooling was proved an effective means for diminishing solvent evaporation during sample loading and minimized the analytical errors involved in Chl a measurements with a fluorescence microplate reader. A set of operating parameters (settling time, detector gain, sample volume) were also optimized to further improve the intensity and reproducibility of Chl a fluorescence signal. A quadratic regression model provided the best fit (r 2=0.9998) across the entire calibration range (0.05-240pgμL-1). The method offered excellent intra- and interday precision (% RSD 2.2 to 11.2%) and accuracy (% relative error -3.8 to 13.8%), while it presented particularly low limits of detection (0.044pgμL-1) and quantification (0.132pgμL-1). The present assay was successfully applied on marine phytoplankton extracts, and the overall results were consistent (average % relative error -14.8%) with Chl a concentrations (including divinyl Chl a) measured by high-performance liquid chromatography (HPLC). More importantly, the microplate-based method allowed the analysis of 96 samples/standards within a few minutes, instead of hours or days, when using a traditional cuvette-based fluorometer or an HPLC system. Graphical abstract TChl a concentrations (i.e. sum of Chl a and divinyl Chl a in ng L-1) measured in seawater samples by HPLC and fluorescence microplate reader.

Characterization of growth inhibition of oral bacteria by sophorolipid using a microplate-format assay

Sophorolipid (SL) is a class of glycolipid biosurfactant produced by yeast and has potent antimicrobial activity against many microorganisms. In this paper, a microplate-based method was developed to characterize the growth inhibition by SL on five representative species of caries-causing oral bacteria. Bacterial growth on microplate in the absence and presence of varying concentrations of SL was continuously monitored by recording the absorbance at 600nm of the cultures using a microplate reader. The results showed that SL completely inhibited the growth of the Lactobacilli at ≥1mg/ml and the Streptococci at much lower concentrations of ≥50μg/ml. More importantly, we further defined the mechanism of antimicrobial activity of SL by analyzing the pattern of the cell growth curves. SL at sublethal concentrations (<1mg/ml) is bactericidal towards the Lactobacilli; it lengthens the apparent cell-doubling time (Td) and decreases the final cell density (as indicated by A600nm) in a concentration-dependent manner. Against the oral Streptococci, on the other hand, SL at sublethal concentrations (<50μg/ml) is bacteriostatic; it delays the onset of cell growth in a concentration-dependent fashion, but once the cell growth is commenced there is no noticeable adverse effect on Td and the final A600nm. Scanning electron microscopic (SEM) study of L. acidophilus grown in sublethal concentration of SL reveals extensive structural damage to the cells. S. mutans grown in sublethal level of SL did not show morphological damage to the cells, but numerous protruding structures could be seen on the cell surface. At the respective lethal levels of SL, L. acidophilus cells were lysed (at 1mg/ml SL) and the cell surface structure of S. mutans (at 130μg/ml SL) was extensively deformed. In summary, this paper presents the first report on a detailed analysis of the effects of SL on Lactobacilli and Streptococci important to oral health and hygiene.

Microplate-Based Detection of Lytic Polysaccharide Monooxygenase Activity by Fluorescence-Labeling of Insoluble Oxidized Products.

Most existing methods for screening the activity of lytic polysaccharide mono-oxygenases (LPMOs) on polysaccharides are based on the detection of soluble oxidized sugars. This approach might underestimate the total performance of LPMOs since oxidation events that do not lead to oligosaccharide release are not detected. Using PcLPMO9D as a model enzyme, a microplate-based method has been developed to detect C1-oxidizing LPMO activity by covalently linking a water-soluble fluorophore to oxidized positions within the cellulose fiber. This fluorescence method was validated using X-ray photoelectron spectroscopy and then combined with high-performance anion-exchange chromatography to track total PcLPMO9D activity.